1. Field of the Invention
The present invention refers to the use of quinone Q10 (ubiquinone) as active principle to be used in a pharmaceutical composition for ocular topical use for the treatment of ophthalmologic pathologies and for the prevention of the undesired side-effects in the cornea, following a treatment of refractive surgery with excimer laser and exposition to ultraviolet radiation of solar light and other sources. By way of example, under refractive surgery the photorefractive keratectomy (PRK) and the laser-assisted in situ keratomileusis (LASIK) are to be meant.
2. Background of the Invention
In the last few years a new type of surgical technique, the photorefractive keratectomy (PRK) correcting the refractive vices, such as myopia, hypermetropia and astigmatism by the use of an excimer laser has become popular and spread. This surgical procedure provides a first step of corneal disepithelization which allows, even if with different techniques, the removal of the first corneal layer which is the epithelium and the exposure of the underneath corneal stroma.
The excimer laser acts through a photoablative action exactly at the level of the frontal stromal surface by causing the remodelling thereof. This involves in the last analysis a remodelling of the frontal corneal surface since the epithelium reforming during the first days after operation follows the profile of the photoablated frontal stromal surface.
The problems connected to the PRK are represented by the possible undesired side-effects reducing the possible regression of the refractive outcome and, or, the formation of a small corneal haze which, if present in great quantity, causes a serious quantitative and qualitative decay in vision functionality, in this case not corregible even with glasses. The regression and the haze have both an etiology due to a plurality of factors.
The first factors are of individual character (genetic predisposition) and as such are not influenceable. The type of photoablative mechanism and the size of the photoablated area are then important; wider and more regular ablation areas, in fact, seem to increase the stability of the refractive outcome. The improvement of photoablative technique, however, is related to the excimer laser technique.
The last important aethiologic factor is linked to the apoptosis role. (Wilson S E, et al., Exp. Eye Res., 1996,62:325-328; Helena B C, Inv. Ophthal. and Visual Science, 1988,39:276-283).
The apoptosis is a programmed cell death which, contrary to usual necrotic processes, is accompanied by a poor inflammatory response and by a failure in releasing the cell degradation components which otherwise would cause damage to adjacent tissue. In the cornea an apoptosis of stromal keratocytes has been observed both following herpes simplex infections, and in response to an epithelial insult such as the one performed in a photorefractive keratectomy operation during the first corneal disepithelization step. This disepithelization, whether it occurs by mechanical action, or by other technique, involves the release of cytokines (for examples interleukin-1) by damaged epithelial cells, which bond to underneath stromal keratocytes by then mediating the apoptosis thereof. The programmed cell death of these stromal keratocytes involves an activation of the adjacent keratocytes aiming at repopulating the frontal stroma and it is associated to an increased deposition of collagen and to a disorganization thereof, both phenomena considered responsible for the haze appearance and for the regression after photorefractive keratectomy operation (for a review see Wilson S E, J. Refractive Surgery, 1997, 13:171-175). The apoptosis role in a wide range of ocular pathologies has been widely demonstrated (see Capaccioli S. et al., in Bisantis C. and Carella G. xe2x80x9cVascular systems of the optic nerve and perioptic area.xe2x80x9d I.N.C Editor, Rome, Italy, 1998)
As it is known, the agents triggering the apoptosis are various and can be of chemical (for example genotoxic drugs), physical (radiations, mechanical insult) or biological (for example virus) nature. (Capaccioli et al., in: xe2x80x9cMonografie della Società Italiana di Oftalmologiaxe2x80x9d, Publishing house I.N.C., Rome, 1998).
As far as the ultraviolet radiation is concerned, it is ascertained by now that, in refractive keratectomy-operated patients, they induce at corneal level oxidizing processes involved both in forming xe2x80x9chazexe2x80x9d and regression. In fact, in PRK-and-LASIK-operated patients, there is often the detection of a higher haze incidence at the end of the summer season, that is at the end of that year period wherein the exposure to solar radiation is maximum. These oxidizing processes involve the release of free radicals already demonstrated in laboratorial preparations of ephitelium of test animals treated with excimer laser, free radicals which are potentially able to trigger the apoptosis process and direct cell damage.
The quinone Q10 plays an essential role in nature since it belongs to the mitochondrial transportation chain of electrons and it is known as an effective antioxidant.
The problem the present invention is based upon is then to provide a drug to oppose the apoptosis of corneal stromal keratocytes in the photorefractive keratectomy (PRK and LASIK), as well as to reduce the oxidizing processes induced by exposition and ultraviolet radiation of the solar light.
Therefore, it is an object of the present invention the use of the ubiquinone Q10 coenzyme, in the form of collyrium for topical ophthalmic use, to manufacture a drug for the treatment of ocular pathologies in general and, in particular, effective in the prevention and treatment of corneal haze following to corneal trauma, general surgery and refractive surgery; to prevent the regression of corrective effects after refractive surgery performed by conventional surgery or by laser radiation; to protect the eye against damage determined by radiation of solar light and by ultraviolet radiation.
Furthermore, the topical ophthalmic use of the ubiquinone Q10 for the pathology prevention and treatment, or for incidental or post-surgery trauma, of the camera frontal bulbi, including iris and crystalline, is included in the scope of the invention. More particularly, it is another object of the present invention a formulation in the form of collyrium and a process for the preparation thereof for ophthalmical administration of ubiquinone, for the cornea protection against the apoptosis of corneal stromal keratocytes which would trigger following treatment of refractive and/or excimer laser surgery and exposure to solar ultraviolet radiation.
It is known that apoptosis is a phenomenon of programmed cell death and, as such, characterized by very precise signalling routes inside the cell. Despite fundamental events have been up to now detected and grouped into virtual operating compartments (initiator, modulator, effector compartment) the precise molecular mechanisms being at the base thereof are extremely complicated, and the understanding thereof is up to now widely full of gaps. It is sure that whereas the modulator and effector compartment may be linked to a limited number of alternative signalling routes, the initiator compartment responds to a plurality of stimuli quite different among them, even if all of them are of potentially apoptotic nature. Among them, biological agents, including the virus, the hypoxia and a variety of chemical and physical agents including genotoxic agents, oxidant agents, exciting, ionizing and electromagnetic radiations, mechanical insults, stimulus by kitocynes, defect in trophic factors, etc., have been detected.
Even if the Q10 antioxidant properties have been well known for some time, the characterization thereof as agent having influence on the apoptosis of corneal stromal keratocytes in the photorefractive keratectomy is not known in the current state of art. In fact, there is exclusively indirect evidence about a Q10 involvement in the apoptosis mechanism. For example, if vehiculated in the serum, the Q10 has resulted to bring benefit in the therapy of reperfusion ischemia insult, a pathology wherein the apoptosis is well-known involved (Sharov et al., Am J. Pathol. 148 (1):141-9, 1996). However, in this pathology it is believed that the Q10 role is also to maintain integral the electrons transport chain in order to produce an ATP quantity sufficient for an optimum cardiac activity. Therefore, the Q10 role would be generally considered not only as an antioxidant molecule but also as key component of the respiratory chain, wherein this coenzyme participates as component of even three multienzymatic complexes, process which is reflected in the production of chemical energy in the form of ATP.
A third role the ubiquinone has recently resulted to play is to regulate the so-called micropore of mitochondrial permeability transition (Permeability Transition Pore, PTP) present in the inner mitochondrial membrane, wherein the Q10 inserts by bonding to a specific bonding place thereof. The functional status of the above-mentioned micropore is regulated by the complex I of the respiratory chain the Q10 is part of. At this level the Q10 acts by inhibiting the micropore opening, an early event of the apoptotic programme since it enables the cytochrome emission into the cytoplasm and the bonding thereof to APAFI which triggers the caspase activation process (Fontaine E. et al. J. Biol. Chem. 271:6746-6751, 1998).
What has been above described does not enable to assert that what has been observed in the cardiac pattern is tout court extendible to the corneal pattern.
Hereinafter the only known work is reported wherein the Q10 is directly associated to a molecule involved in the signalling route of the apoptotic process (Barroso M. P. et al, J. Bionerg. Biomembr., 1997, 29:259-67). In fact, it seems that the Q10 present in the plasmatic membrane decreases the ceramide levels, degradation product of the sphyngomyelin and known transducer of the apoptotic signal.
By referring to the PRK, even if it has been ascertained that the undesired effects of the technique are largely due to the apoptotic death of cells, the immediate effects of excimer laser on/the cytoxicity mechanisms thereof are not known.
It has been demonstrated that other antioxidants such as the ascorbic acid, the pyrrolidinditiocarbamate (PDTC), the Vitamin E, and likes, have contrasting effects on the apoptosis induced by various stimuli. For example the ascorbic acid has demonstrated to be effective in the inhibition of the apoptosis induced by oxidant stress with quite high concentrations, (Witenberg B et al., Biochem. Pharmacol., 1999, 57:823-32) but the derivatives thereof have demonstrated to be toxic in presence of H2O2 (Iwasaka K., Biochem. Anticancer Res., 1998, 18:4333-7).
The PDTC has resulted to protect against apoptosis induced by Tumor Necrosis Factor (TNF) (Higuchi M., et al. Oncogene, 1998, 17:2515-2524), but, as Vitamin E, it has resulted to induce apoptosis in a cellular line of the rectum cancer (CRC) (Chinery R, et al. Nat. Med. 1997, 11:1233-41).
Therefore, based upon the antioxidant features thereof only, it could not be foreseen that the use of Q10 as preventive and therapeutic agent in the corneal refractive surgery could have an advantageous effect in the prevention and treatment of the undesired side-effects in the photorefractive surgery (PRK and LASIK) and in the exposure to ultraviolet radiation.
Similarly, it could not be foreseen to which extent the use of Q10 may protect cells against apoptosis in those ophthalmologic diseases wherein this cell death process seems to play a key role in the pathogenetic mechanisms.
On the other side, the fact that for the Q10 there are no data about cytotoxic effects of the concentrations up to now utilized in vitro researches, is in favour of the drug harmlessness with respect to toxic effects.
It has been experimented in laboratory the Q10 protective effect against apoptosis and necrosis induced by ultraviolet radiation (both with 193 nm ArF excimer laser and with 254 nm radiation) on rabbit corneal keratocyte cultures. The apoptosis has been evaluated by means of early and late markers such as analysis of the cytoplasm redox status (malonaldehyde assay), ATP levels, confocal microscopy and transmission electronics, gene p53 expression, qualitative/quantitative analysis of cell morphological changes by means of videomicroscopy at intervals.
The doses to be utilized should be in the range between 2 xcexcM and 500 xcexcM, preferably 10 xcexcM.
Furthermore, a preparation acceptable from the pharmaceutical point of view for the topical administration as Q10 collyrium has been devised